New Direction

Epigenetics describes the mechanisms by which chromatin-associated
proteins and post-translational modifications regulate gene expression.
These heritable traits, not linked to changes in the DNA sequence, refer
to the epigenetic code or pattern of post-translational modifications
on proteins and DNA that lead to the regulation of transcription (1).
The role of the epigenetic regulators and transcription factors in
maintaining the genome in open or closed conformation, thereby
controlling the transcriptional programme, is fundamental to cellular
processes such as proliferation, development and differentiation.
Dysregulation of epigenetic mechanisms is closely linked to the
progression of disease.
Readers, Writers and Erasers

Epigenetic
control over transcriptional programmes facilitates appropriate and
specific cellular function within each tissue and organ system, in spite
of a common genomic sequence shared by every non-germline cell in the
body. Adding (writing) and removing (erasing) post-translational
modifications – for example, acetylation, methylation and ubiquitination
– to the protein components of chromatin is followed by ‘reading’,
which dictates gene expression and eventual phenotypic response (2,3).
Through this mechanism, cellular programmes are tightly regulated, yet
responsive to stimuli.

Inappropriate epigenetic activity can
lead to cellular dysfunction, and is implicated in a variety of
diseases, such as cancer and autoimmune disorders; epigenetics is
therefore a promising avenue for research. The first of these therapies
to reach the clinic have been histone deacetylase and DNA
methyltransferase inhibitors (4). They have been approved for the
treatment of cutaneous T cell lymphoma and myelodysplastic syndromes,
and continue to be investigated in numerous drug development programmes
targeting writers and erasers.

The development pipeline is also
starting to fill with drugs targeting readers, or the proteins that
recognise the pattern of post-translational modifications and guide gene
expression. Readers – such as those within the bromodomain class of
proteins – recognise and bind acetylated lysine residues on histone
tails, found in actively transcribing regions of chromatin (5), and
serve as a docking platform for the assembly of large transcriptional
complexes and the recruitment of key transcriptional proteins, like
positive transcription elongation factor (p-TEFb) (6). This
bromodomain-containing family, consisting of approximately 46 diverse
nuclear and cytoplasmic proteins, has been the focus of significant drug
development efforts in the oncology, autoimmune and vascular disease
arena (7).

BET Proteins

Bromo and extraterminal
(BET) proteins make up a sub-group of the 46 bromodomain-containing
proteins and consist of four members: BRD2, BRD3, BRD4 and BRDT. All
four BET proteins contain two conserved N-terminal bromodomains (BD1 and
BD2), which recognise and bind acetylated lysine residues on histone
tails and other nuclear proteins (8). These interactions localise BET
proteins to discrete locations along the chromosome, where they recruit
and facilitate assembly of factors to influence gene expression (9). BET
proteins regulate genes that play a part in proliferation, cell cycle
progression and apoptosis (9,10).

Dysfunction of these proteins
has been associated with the development of aggressive tumours. Notably,
nuclear protein in testis (NUT) midline carcinoma (NMC) arises as a
result of a fusion protein between the N-terminal bromodomain of BRD3 or
BRD4 with NUT. In the first pivotal studies in this space, a BET
inhibitor, JQ1, was shown to bind to and target the bromodomain aspect
of this fusion protein responsible for driving the oncogenic
transformation in the tumour (11). By way of its inhibition, the fusion
protein was released from chromatin, shutting down the oncogenic gene
expression and validating BET proteins' roles in transcriptional
programmes.

By recognising chromatin conformation
(post-translational modifications), BET proteins can contribute to
pathological gene expression. This role is even more magnified in states
of aberrant lysine acetylation, where chromatin is in a constitutively
open, transcriptionally available conformation. Reading by BET and other
bromodomain-containing proteins can, therefore, contribute to this
inappropriate gene expression, as has been demonstrated with genes such
as c-Myc (12) and Aurora B (13). Importantly, based on findings showing
the presence of BET proteins at super-enhancer sites driving the
expression of oncogenes, regulating the function of these readers is an
attractive strategy for shutting down expression in a variety of cancers
(14). Consequently, BET inhibition is a valid therapeutic strategy in
oncology, as well as other states of dysregulated gene expression (15).

Clinical Studies in Cancer

Findings
from preclinical studies have fuelled interest in the field of
epigenetic drug development, with sights set on new and improved small
molecules for BET inhibition. Based on the protein-protein binding
between BET proteins and acetylated lysines present in the chromatin,
small-molecule inhibitors are being avidly pursued. Notably, several
different, orally available, low molecular weight scaffolds and
molecules have been developed – a number of which have moved into the
clinic and are in early clinical trials, including:

I-BET762/GSK525762A
– Phase 1 in relapsed, refractory haematologic malignancies, as well as
NUT midline carcinoma and other cancers (clinicaltrials.gov
identifiers: NCT01943851 and NCT01587703)

Based
on their function in binding acetylated residues on histones and other
proteins, and facilitating assembly of transcriptional complexes,
bromodomain-containing proteins play a role in regulating transcription.
This regulation extends beyond oncogenes and modulates a variety of
cellular programmes inherent to correct cell functioning and
environmental response. For instance, regulation of the transcriptional
programme upstream of pro-inflammatory mediators is one of the prime
mechanisms governing inflammatory diseases and states. NF-κB is a key
regulator of the immune response, and its part in directing
transcription of downstream mediators involves the recruitment of NF-κB
co-activators, such as BRD4 (15,16). It is therefore not surprising that
BET inhibitors have been shown to potently suppress the inflammatory
transcriptional response.

A recent study by Brown et al
has extended these observations by investigating the genome-wide
relationship between these two factors in endothelial cells (17). The
findings demonstrated that a subset of NF-κB functions occur through
super-enhancer complexes, which can be influenced by BET inhibition. In
fact, JQ1 administration significantly attenuated disease progression in
a mouse model of aberrant inflammation (in atherosclerosis), thus
inferring the important role of BET proteins, and the potential for BET
inhibition in diseases with an inflammatory component, such as
atherosclerosis, sepsis or autoimmune diseases (15,18).

Beyond
effects on NF-κB, BET inhibition has demonstrated a role in the
differentiation and activation of TH17 cells – as well as the
pro-inflammatory functions of TH1 cells – and likely has a role in other
immune responses (18,19). BET proteins have also been identified as
central co-activators of transcription factors driving aberrant gene
expression in heart failure and pathological remodelling of the heart.
Treatment with BET inhibitors arrested hypertrophy and heart failure in
murine models, providing a rationale for developing these inhibitors as
therapeutic agents in heart disease (20). BET family members have
additionally been implicated in the regulation of viral genome
replication and expression of viral proteins. This holds therapeutic
potential in the activation of latent viruses such as HIV (21). In doing
so, the awakened virus can be targeted with antiviral agents, making
BET bromodomain inhibition a viable strategy for addressing and
eradicating HIV.

Upstream of many transcriptional programmes,
BET inhibition may be a new frontier for regulating and modulating
multiple cellular biologies simultaneously, which could provide
therapeutic benefit for many diseases.
BETs in Other Indications

The
most advanced BET inhibitor in clinical development is RVX-208. This
compound was originally discovered in a screen for Apolipoprotein A1
(APOA1) mRNA inducers in hepatocyte cell cultures, and has been shown to
raise APOA1 and high-density lipoprotein (HDL) in humans (22). RVX-208
binds preferentially to the second bromodomain of BET family members,
with a 20-fold or higher selectivity for the second bromodomains of
BRD2, BRD3 and BRD4 versus the first bromodomain (23,24). This
inhibition modulates expression of a variety of genes including APOA1 –
the core protein component of HDL (15,23,24). Elevating APOA1 increases
reverse cholesterol transport, which is essential for atherosclerotic
plaque regression in the treatment of high-risk cardiovascular disease
patients. RVX-208 has been shown to raise the body’s de novo
synthesis of APOA1 and HDL by this mechanism in a number of Phase 1 and 2
clinical trials (25), in indications related to cardiovascular disease
(CVD) and metabolic disease (clinicaltrials.gov identifiers:
NCT01728467, NCT01067820, NCT01423188, NCT01058018 and NCT00768274).

In
post hoc analysis of the SUSTAIN and ASSURE trials, pooled data
analysis demonstrated statistically significant increases in APOA1 and
other markers of reverse cholesterol transport (26). Beneficial effects
were also observed on CVD biomarkers, such as high-sensitivity
C-reactive protein, alkaline phosphatase, and components of the
complement and coagulation cascades. RVX-208 treatment also had an
effect on the incidence of major adverse cardiovascular events (MACE);
defined as composite of death, non-fatal myocardial infarction, coronary
revascularisation procedures, and hospitalisation for unstable angina
or heart failure. Data from this analysis demonstrated a 55% relative
risk reduction (RRR) in the incidence of MACE in CVD patients, and a
more pronounced 77% RRR in patients with a history of diabetes mellitus.
Taken together, the combined effects of BET inhibition on multiple
biologies known to impact vascular risk may, in part, explain the
reduction in MACE events – a promising finding for this class of
compounds.

Based on its mechanism of action, BET inhibitors such
as RVX-208 may have additional utility in indications related to its
effects on multiple biologies – for example, chronic kidney disease,
Alzheimer’s disease, heart failure, peripheral artery disease and other
immune-related disorders.
New Paradigm

Epigenetics
is a novel and promising area of research with the potential to
simultaneously modulate multiple biologies. Molecular and mechanistic
studies have already provided exciting insight into the function of BET
inhibitors, and indicate a possible shift from the ‘one target-one drug’
development model, to a more physiologically relevant approach of
concurrently modulating multiple biological processes, all of which
contribute to the diseased state. BET inhibitors may therefore be at the
forefront of this new paradigm for drug development.

Ewelina Kulikowski is Vice President of Scientific Development at Resverlogix Corp. She oversees various aspects of the scientific, clinical and business development of RVX-208 and other novel drugs for the treatment of CVD, neurodegenerative disorders and inflammatory diseases. Ewelina received her PhD in Oncology from the University of Calgary, Canada, and has been at Resverlogix since 2005.

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